Compared to skin, relatively little is known about the basic biology of prostate epithelial stem cells, including their precise anatomical location and how the stem cell niche boundaries are defined. Prostate stem cells have been defined as cells that are able to survive the involution of the prostate and the apoptotic loss of the luminal layer that follows castration or acute androgen withdrawal, and as cells able to reconstitute the luminal layer and regenerate the prostate after androgen restoration. The presence of intermediate cells that contain both basal and luminal cell markers and that may represent transit amplifying cells may support these lineage relationships. However, several recent findings call into question the assumption that prostate stem cells are restricted to the basal layer and also raise questions about the lineage relationship between basal cells and luminal cells. Using techniques that we have previously used to characterize the location and lineage relationships of keratinocyte stem cells in the skin, we would like to characterize the anatomical location of prostate stem cells in the prostate elucidate the lineage relationships between the basal and luminal cell layers. Transgenic mouse models that will selectively express a histone-GFP (H2B-GFP) fusion protein in different prostate epithelial layers in a tet-regulatable manner have been created. These mice will be used along with BrdU-labeling studies to determine the anatomical location of prostate stem cells as well as lineage relationships between the basal and luminal epithelial layers. Another significant problem for characterizing prostate stem cells is the absence of standard in vivo assays to grow and propagate human prostate cancer and normal prostate tissue, a critical requirement for assessing the stem cell behaviors of self-renewal and long-term tissue repopulation. We are currently developing 3-dimensional in vitro and in vivo (sub-cutaneous) assays that are able to reconstitute a normal prostate or recapitulate prostate cancer in an in vivo animal model, analogous to the manipulations that we currently perform with normal skin cells. To date, we have been studying primary prostate epithelial and stromal cells (derived from fresh prostate tissue) and immortalized cell lines derived from prosate cancers in these in vitro and in vivo model systems.